Mitochondria are the powerhouse of eukaryotic cells such as cardiomyocytes. Besides their role in free energy transduction, they are responsible for maintaining intracellular ionic homeostasis (e.g., Ca2+, Na+, Mg2+, H+, K+) and handling toxic byproducts of energy metabolism (e.g., ROS). Consequently, functional defects in these processes can lead to mitochondrial dysfunction, cell death, and pathogene- sis of several diseases. A quantitative understanding of regulation of mitochondrial function through the kinetics of mitochondrial cation transport and buffering and ROS generation and scavenging will be cru- cial in developing a systems-based, engineering understanding of mitochondrial, cellular, whole-organ, and whole-organism function and associated disease processes. The overall goal of this collaborative proposal is therefore to quantify the biophysics and chemical kinetics associated with mitochondrial handling of pH, cations, and ROS in cardiomyocytes and to understand how they individually and col- lectively influence mitochondrial function. We hypothesize that cations selectively inhibit or activate specific cation channels and exchangers to regulate trans-matrix fluxes and intra-matrix concentrations of these cations. Furthermore, the intra-matrix free concentrations of cations (e.g., Ca2+) are governed by a dynamic buffering mechanism due to dynamic binding of the cations with phosphates (ATP, ADP, PI) and substrates (TCA cycle intermediates) during transient respiration, which in turn regulate mito- chondrial function, including the rate of ROS generation. Pathophysiological and chronic alterations in these mechanisms can lead to mitochondrial dysfunction and cellular injury. These hypotheses will be tested through an iterative process between experimental measurements and computational modeling of mitochondrial bioenergetics and electrophysiology (changes in trans-matrix cation concentrations and fluxes, NADH and FAD redox states, membrane potential, respiration, and ROS production with normal and abnormal perturbations in extra-matrix cation concentrations) in both isolated cardiac mito- chondria and isolated intact hearts, with the refinement of both the experimental design and computa- tional model as the research unfolds. The model will provide the basis for improving the experimental design and specific hypotheses; in turn, the experimental data will provide the basis to refine and ex- tend the model. By the use of this integrative approach we will also be able to understand regulation of ROS production during ischemia and reperfusion (I/R). Recovery of cardiac function after I/R is critically dependent on rapid recovery of mitochondrial function to restore ATP production, which prevents de- rangement of cytosolic and mitochondrial cation homeostasis and avoid cellular injury or death. The proposed combined approach represents a novel strategy in understanding the integrated function of cardiomyocytes under physiological and pathophysiological conditions.